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Administrative data

Description of key information

The test item is not considered to possess an irritant potential to skin (reference 7.3.1-1).


The test item did not show an eye irritating potential (reference 7.3.2 -1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-10-14 to 2020-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
31 July 2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
June 18, 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Justification for test system used:
The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17 (from Episkin/SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 20-RHE-133
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 2020-10-14
- Expiry date: 2020-10-19

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: minimum volume of 25 mL DPBS was used
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes)
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Acceptance Criteria: OD >0.7; Result: OD 1.3 (CV = 7.7 %)
- Barrier function: Acceptance Criteria: 4.0 h - Morphology: Acceptance Criteria: Number of cell layers > 4, Multi-layered, highly differentiated epidermis consisting of basal, spinous and granulär layers, and a multi-layered Stratum corneum; Result: 6.5 cell layers, Satisfactory
- Contamination: not specified
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i. e. the test item is not a direct MTT reducer. Therefore, no additional control was needed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the viability is less than or equal to 50 %.
- The test substance is considered to be non-corrosive and non-irritant to skin if the viability is greater than 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 mg ± 2 mg per tissue (10 µL of deionised water used to wet tissue before application)

NEGATIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue

POSITIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue
- Concentration: 5 %
Duration of treatment / exposure:
42 minutes (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
76.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
1.6
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i. e. the test item is not a direct MTT reducer.
- Colour interference with MTT: The test item has no colorant properties. Therefore, no interference was observed.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values: Please refer to "Any other information on results".

Table 1: Study results






















































































Substance



Optical Density (OD)



Viability (%)



Negative Control
1x PBS



1



1.73



99.4



2



1.812



104.1



3



1.679



96.5



mean



1.74



100



standard deviation (SD)



3.8



Positive Control
SDS (5 % aq.)



1



0.027



1.6



2



0.026



1.5



3



0.027



1.6



mean



0.027



1.6



standard deviation (SD)



6.3



Test item



1



1.261



72.5



2



1.326



76.2



3



1.406



80.8



mean



1.331



76.5



standard deviation (SD)



5.5



 


Table 2: Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data
















 



Acceptance Criterion



Result



Negative control OD



≥0.8 and ≤ 3.0



1.679 to 1.812



 


Table 3: Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories


























 



Acceptance Criterion



Result



Mean OD negative control



≥ 1.2



1.74



Mean viability positive control



< 40 %



1.6 %



SD of group-mean value



≤ 18 %



6.3 % (positive control)


3.8 % (negative control)



 


Table 4: Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory





















 



Acceptance Criterion



Result



Mean OD negative control



≥ 1.415



1.74



Mean viability positive control



≤ 2.72 %



1.6 %



 


Table 5: Test Item Data Acceptance Criteria
















 



Acceptance Criterion



Result



SD of group-mean value



≤ 18 %



5.5 %



 


Table 6: Historical Data (of all tests performed in laboratory)






















Positive control



Negative control



Mean viability [%]



1.42



Mean adsorption [OD570]



1.937



Standard deviation



0.44



Standard deviation



0.26



 

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin.
Executive summary:

The objective of the study conducted according to OECD 439 was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control and the positive control were met. Following treatment with the test item, the tissue viability was 76.5 % and, thus, higher than 50 %, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-12-01 to 2020-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: EU Method B.69 (RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RhCE) TEST METHOD FOR IDENTIFYING CHEMICALS NOT REQUIRING CLASSIFICATION AND LABELLING FOR EYE IRRITATION OR SERIOUS EYE DAMAGE)
Version / remarks:
31 July 2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
June 18, 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.
- Description of the cell system used, and the mycoplasma status of the cell live: The EpiOcular™-model is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. Long term antibiotic and antimycotic free culture with no contamination was applicable (result: sterile). Therefore, the QA statement from the supplier considered a pass for the the sterility criterion.
- Cell line used, its source, passage number and confluence of cells used for testing: Keratinocyte strain: 4F1188; passage number: not specified; Barrier
function: 12.93 min (within acceptance criteria of supplier)
- RhCE tissue or hCE cell construct used, including batch number: EpiOcular™ Tissue (OCL-200, OCL-212) from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot no: 30687
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg

Negative and positive control
- Amount applied: 50 µL
Duration of treatment / exposure:
6 hours (± 15 minutes) at 37 °C
Duration of post- treatment incubation (in vitro):
25 minutes (± 2 minutes) at room temperature and 18 hours (± 15 minutes) at 37 °C
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used:
The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i. e. the test item is not a direct MTT reducer and the test item has no colorant properties. Therefore, no further control was needed.
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: Spectrophotometer (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60 %. If the mean percent tissue viability is less than or equal 60 %, no prediction can be made.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
The results are acceptable if:
The negative control OD is >0.8 and <2.5.
The mean relative viability of the positive control is:
a) 30-minute exposure (treatment of liquid test items): below 50 % of control viability
b) 6-hour exposure (treatment of solid test items): below 50 % of control viability
- Reference to historical data of the RhCE tissue construct: Tissue viability via MTT QC assay, 1 hour, n=3; Acceptance criteria of OD (540-570 nm) [ 1 . 1 - 3 . 0] with a result of 1.899 ± 0.045 for the batch according to supplier
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: not specified
- Acceptable variability between tissue replicates
The difference of viability between the two relating tissues of a single chemical is <20 % in the same run (for positive and negative control tissues and tissues of single chemicals).
Irritation parameter:
mean percent tissue viability 
Value:
106.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
39.7
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative control OD is >0.8 and <2.5 (1.732 and 1.686).
- Acceptance criteria met for positive control: yes. The mean relative viability of the positive control is below 50 % of the negative control viability (39.7 %).
- Other: The difference of viability between the two relating tissues of a single chemical is <20 % (values between 0.0 % to 5.1%) in the same run (for positive and negative control tissues and tissues of single chemicals).
























































































SubstanceOptical Density (OD)Viability (%)
Negative Control11.732101.3
water21.68698.7
 mean1.709100
 standard deviation (SD)1.84
 Difference between tissue replicates2.6
Positive Control10.67939.7
SDS (5 % aq.)20.67939.7
 mean0.67939.7
 standard deviation (SD)0
 Difference between tissue replicates0
Test item11.782104.3
21.87109.4
mean1.836106.9
standard deviation (SD)3.61
Difference between tissue replicates5.1
Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not show an eye irritating potential.
Executive summary:

The objective of the study conducted according to OECD 492 was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. After treatment with the negative control (sterile deionized water) the OD was 1.732 and 1.686 of the two treated tissue, respectively (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 39.7 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 106.9 % and, thus, higher than 60 %, i.e. according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation


The objective of the study conducted according to OECD 439 was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control and the positive control were met. Following treatment with the test item, the tissue viability was 76.5 % and, thus, higher than 50 %, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).


 


Eye irritation


The objective of the study conducted according to OECD 492 was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. After treatment with the negative control (sterile deionized water) the OD was 1.732 and 1.686 of the two treated tissue, respectively (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 39.7 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 106.9 % and, thus, higher than 60 %, i.e. according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008


The available data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be require no classification for eye irritation or damage and skin irritation or corrosion under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.

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